CN102770536B

CN102770536B - Mutant enzyme and application thereof

Info

Publication number: CN102770536B
Application number: CN201080055012.4A
Authority: CN
Inventors: 西尾享一; 小池田聪
Original assignee: Amano Enzyme Inc
Current assignee: Amano Enzyme Inc
Priority date: 2009-12-05
Filing date: 2010-11-22
Publication date: 2014-04-30
Anticipated expiration: 2030-11-22
Also published as: CN103981158A; CN102770536A; US8883434B2; KR101814588B1; JPWO2011068050A1; EP2508600B1; JP5846687B2; KR20120099043A; WO2011068050A1; JP2016034280A; JP6059784B2; US9458434B2; US20150024461A1; EP2508600A4; EP2508600A1; US20120244565A1

Abstract

Disclosed is a novel enzyme that exhibits glucose dehydrogenase activity. Furthermore, disclosed is a novel method pertaining to enzyme modification. The mutant enzyme comprises an amino acid sequence wherein one or at least two amino acids selected from the group consisting of (1)-(13) below are replaced with another amino acid in the amino acid sequence of a microorganism-derived glucose oxidase: (1) the amino acid corresponding to the amino acid at position 115 of the amino acid sequence indicated by SEQ ID NO: 1; (2) the amino acid corresponding to the amino acid at position 131 of the amino acid sequence indicated by SEQ ID NO: 1; (3) the amino acid corresponding to the amino acid at position 132 of the amino acid sequence indicated by SEQ ID NO: 1; (4) the amino acid corresponding to the amino acid at position 193 of the amino acid sequence indicated by SEQ ID NO: 1; (5) the amino acid corresponding to the amino acid at position 353 of the amino acid sequence indicated by SEQ ID NO: 1; (6) the amino acid corresponding to the amino acid at position 436 of the amino acid sequence indicated by SEQ ID NO: 1; (7) the amino acid corresponding to the amino acid at position 446 of the amino acid sequence indicated by SEQ ID NO: 1; (8) the amino acid corresponding to the amino acid at position 472 of the amino acid sequence indicated by SEQ ID NO: 1; (9) the amino acid corresponding to the amino acid at position 511 of the amino acid sequence indicated by SEQ ID NO: 1; (10) the amino acid corresponding to the amino acid at position 535 of the amino acid sequence indicated by SEQ ID NO: 1; (11) the amino acid corresponding to the amino acid at position 537 of the amino acid sequence indicated by SEQ ID NO: 1; (12) the amino acid corresponding to the amino acid at position 582 of the amino acid sequence indicated by SEQ ID NO: 1; (13) the amino acid corresponding to the amino acid at position 583 of the amino acid sequence indicated by SEQ ID NO: 1.

Description

Mutant enzyme and uses thereof

Technical field

The present invention relates to the modifying method of mutant enzyme and enzyme, glucose oxidase of changing through desaturase (dehydrogenase) and preparation method thereof etc. is provided.The application advocates the right of priority of No. 2009-277096th, Japanese patent application based on December 5th, 2009 application, quotes the full content of this patent application by the mode of reference.

Background technology

Use the simple type oneself blood sugar detection device of electrochemica biological sensor to be used widely.In this biosensor, utilizing the enzyme take glucose as substrate is glucose oxidase (being designated hereinafter simply as " GO "), Hexose phosphate dehydrogenase (being designated hereinafter simply as " GDH ").GO has advantages of that the specificity for glucose is high and excellent heat stability is such, is noted but then following problem: in the mensuration of using GO, be easily subject to the impact of the dissolved oxygen in working sample, dissolved oxygen impacts measurement result.

On the other hand, as be not subject to the impact of dissolved oxygen and at NAD(P) enzyme of glucose being had an effect in non-existent situation, known take pyrroloquinoline quinone (PQQ) below the GDH(of coenzyme, referred to as " PQQ-GDH ") (for example, with reference to patent documentation 1～3).But there are the following problems for PQQ-GDH: easily dissociation from enzyme of (1) PQQ; (2) low for the selectivity of glucose; And (3) are owing to being generally present in membrane component, therefore in its extraction, lock out operation, have difficulties etc.

Except PQQ-GDH, as be not subject to the impact of dissolved oxygen and at NAD(P) enzyme of glucose being had an effect in non-existent situation, below the known GDH(take flavin adenine dinucleotide as coenzyme, referred to as " FAD-GDH ").Up to now, from aspergillus oryzae (non-patent literature 1～4, patent documentation 4) and terreus (patent documentation 5), obtain respectively FAD-GDH.As the general characteristic of FAD-GDH, known: reactivity higher (, the in the situation that of patent documentation 5 disclosed FAD-GDH, being for example 10% left and right of the functionality to glucose to the reactivity of wood sugar) and optimum temperuture high (for example optimum temperuture of patent documentation 4 disclosed FAD-GDH is approximately 60 ℃) for wood sugar.Should illustrate, to improve practicality etc. as object, attempt energetically the modification of enzyme.The document (patent documentation 6～9) as follows of the modification of report FAD-GDH.

Recently, reported and utilized the structure of GO to carry out the three-dimensional arrangement analysis of FAD-GDH, identification is important (non-patent literature 5,6) to substrate for Glu414 and Arg502.

Patent documentation

Patent documentation 1: TOHKEMY 2000-350588 communique

Patent documentation 2: TOHKEMY 2001-197888 communique

Patent documentation 3: TOHKEMY 2001-346587 communique

Patent documentation 4: No. 2007/139013 brochure of International Publication

Patent documentation 5: No. 2004/058958 brochure of International Publication

Patent documentation 6: TOHKEMY 2009-225801 communique

Patent documentation 7: TOHKEMY 2009-225800 communique

Patent documentation 8: TOHKEMY 2009-159964 communique

Patent documentation 9: TOHKEMY 2008-237210 communique

Non-patent literature 1:Studies on the glucose dehydrogenase of Aspergillus oryzae.I.Induction of its synthesis by p-benzoquinone and hydroquinone, T.C.Bak, and R.Sato, Biochim.Biophys.Acta, 139,265-276 (1967).

Non-patent literature 2:Studies on the glucose dehydrogenase of Aspergillus oryzae.II.Purification and physical and chemical properties, T.C.Bak, Biochim.Biophys.Acta, 139,277-293 (1967).

Non-patent literature 3:Studies on the glucose dehydrogenase of Aspergillus oryzae.III.General enzymatic properties, T.C.Bak, Biochim.Biophys.Acta, 146,317-327 (1967).

Non-patent literature 4:Studies on the glucose dehydrogenase of Aspergillusoryzae.I V.Histidyl residue as an active site, T.C.Bak, and R.Sato, Biochim.Biophys.Acta, 146,328-335 (1967).

Non-patent literature 5:2009 year Japanese medicine association annual meeting main idea collection volume: No. 129: page 3: 118 speech exercise question sequence number: 26Q-pm115

Non-patent literature 6:2009 year Japan biochemical meeting conference main idea collection speech exercise question sequence number: 3T5a-5(3P-109)

Summary of the invention

There is favourable part in FAD-GDH compared with PQQ-GDH.On the other hand, FAD-GDH is higher for the reactivity of wood sugar, therefore when measuring wood sugar load test experimenter's blood sugar, exists and is difficult to detect the such problem of correct measured value.In addition,, because optimum temperuture is high, while therefore measuring, cannot bring into play sufficient activity under the low environment of the mensuration equitemperature of cold district.Under this condition determination, need to carry out temperature correction and also easily produce error at measurment.As mentioned above, there is part to be improved in original FAD-GDH, expects the further raising of practicality.One of problem of the present invention is to respond this requirement.The current problem of the present invention is for providing the novel method of modification of Some Related Enzymes.

As the method for obtaining the GDH that practicality is high, roughly have: (1) is by existing GDH(FAD-GDH, PQQ-GDH) method of modifying and (2) method that microorganism etc. is screened.These methods are existing attempt (for example above-mentioned patent documentation 6,7) mostly, and from now on, the possibility relevant to the creation of more effective enzyme will be low.In view of this situation, the present inventor etc. are conceived to the problems referred to above that do not have FAD-GDH to have in glucose oxidase (GO).The homology of the aminoacid sequence of known GO and FAD-GDH is higher.Be conceived to this homology, determine to adopt and give GDH activity to GO, that is, GO is carried out to the such novel method of GDHization.First, select from the GO of aspergillus niger (Aspergillus niger), the aminoacid sequence of its aminoacid sequence and known multiple FAD-GDH is carried out to multiple ratio pair.Then, by utilizing the three-dimensional arrangement data of comparison result and GO, thereby near the amino acid being arranged in the active centre of GO, although retrieve conservative between FAD-GDH (concomitant high), different amino acid between GO and FAD-GDH.Result is that 13 place's amino acid positions are determined.Then, be produced on that these amino acid positions import sudden change and mutant enzyme, study its characteristic, result is assert the active mutant enzyme rising with the ratio (GDH activity/GO activity) of GO activity of GDH.Therefore, determine to analyze the sequence of this mutant enzyme.Thus, rising to GDH activity, i.e. effective 4 place's amino acid positions of GDHization have successfully been determined.This result has been enlightened on the one hand in initial 13 places that determine, except this 4 sentence, the rising of GDH activity is not had to effect.But, be do not have aspect the amino acid position of finding from the detailed comparison of FAD-GDH by GO different, therefore for all the other the 9 places also under cover improvement to other characteristics such as substrate characteristic, coenzyme specificity, temperature stabilities, improve useful possibility.In addition, by also, with two or more, be also improved the possibility of GDH activity, other characteristic.Thus, all the other 9 place's amino acid positions are also valuable, and it is applied flexibly and is expected.

On the other hand, the further result of research, has successfully determined the especially effectively combination of the position of substitution of GDHization.When adopting this combination, substrate specificity is good (reactivity to wood sugar is low) also.In addition, for the most excellent combination, the optimization of attempting replacing (that is, amino acid whose after the most effectively replacing determined), result to GDHization effectively the amino acid after replacement determined, and found to bring the amino acid after the replacement of GDHization completely.In addition confirmed, also excellent (reactivity to wood sugar is low) of substrate specificity of the mutant of complete GDHization.

, also experience is much added or the high situation of possibility of collaborative effect by resultful two seed amino acids sudden changes being combined produce.Therefore, can say and determine that successful 4 place's sudden change object's positions are not limited to separately, also effective to GDHization about its combination.

In addition, for enzyme of the same race, if in view of the similarity of structure (primary structure, three-dimensional arrangement) high, same sudden change produces the high such technology general knowledge of probability of same effect, can say for and aftermentioned embodiment shown in the GO of aspergillus niger between GO, other GO of the in fact very high rugged mould of Buddhist nun (Penicillium amagasakiense) of structural similarity, the mutation method that applicable the present inventor etc. find.

As mentioned above, the present inventor etc. successfully undertaken GDHization by GO, create the active high saltant type GO of GDH.Meanwhile, also successfully determined the effective amino acid position of the sudden change of GO.In gained saltant type GO, what have does not demonstrate the reactivity to wood sugar.That is,, not demonstrating aspect wood sugar reactive, successfully obtain the GDH that exceeds existing FAD-GDH.

Above achievement confirms on the one hand: between two kinds of high enzymes of structural similarity, by aminoacid sequence relatively with enlisting the services of, and utilize near the such method of the three-dimensional arrangement in active centre to the modification of enzyme (particularly, for modifying the enzyme of object, give the characteristic that characteristic that other enzyme has or other enzyme possess under preferred state) effectively.

What below illustrate the present invention is based on above achievement.

[1] mutant enzyme, by being selected from one or more amino acid in following (1)～(13) by other aminoacid replacement in the aminoacid sequence of the glucose oxidase from microorganism aminoacid sequence formed:

(1) amino acid corresponding with 115 amino acids of the aminoacid sequence shown in sequence number 1;

(2) amino acid corresponding with 131 amino acids of the aminoacid sequence shown in sequence number 1;

(3) amino acid corresponding with 132 amino acids of the aminoacid sequence shown in sequence number 1;

(4) amino acid corresponding with 193 amino acids of the aminoacid sequence shown in sequence number 1;

(5) amino acid corresponding with 353 amino acids of the aminoacid sequence shown in sequence number 1;

(6) amino acid corresponding with 436 amino acids of the aminoacid sequence shown in sequence number 1;

(7) amino acid corresponding with 446 amino acids of the aminoacid sequence shown in sequence number 1;

(8) amino acid corresponding with 472 amino acids of the aminoacid sequence shown in sequence number 1;

(9) amino acid corresponding with 511 amino acids of the aminoacid sequence shown in sequence number 1;

(10) amino acid corresponding with 535 amino acids of the aminoacid sequence shown in sequence number 1;

(11) amino acid corresponding with 537 amino acids of the aminoacid sequence shown in sequence number 1;

(12) amino acid corresponding with 582 amino acids of the aminoacid sequence shown in sequence number 1;

(13) amino acid corresponding with 583 amino acids of the aminoacid sequence shown in sequence number 1.

[2] mutant enzyme according to [1] wherein, is the aminoacid sequence of

sequence number

1 or 2 from the aminoacid sequence of the glucose oxidase of microorganism.

[3] mutant enzyme according to [1] or [2], wherein, the amino acid that substituted amino acid is (3), amino acid, the amino acid of (7) or the amino acid of (12) of (5) or be selected from the plural amino acid whose combination in them.

[4] according to the mutant enzyme [3] described, wherein, for the amino acid of (3), amino acid after replacement is L-Ala, and for the amino acid of (5), the amino acid after replacement is L-Ala, for the amino acid of (7), amino acid after replacement is Histidine, and for the amino acid of (12), the amino acid after replacement is Serine, arginine, leucine or proline(Pro).

[5] mutant enzyme according to [1] or [2], wherein, amino acid, the amino acid of (7) or the amino acid of (12) that substituted amino acid is (3) or be selected from the plural amino acid whose combination in them.

[6] according to the mutant enzyme [5] described, wherein, for the amino acid of (3), amino acid after replacement is L-Ala, and for the amino acid of (7), the amino acid after replacement is Histidine, for the amino acid of (12), the amino acid after replacement is Serine, arginine, leucine or proline(Pro).

[7] according to the mutant enzyme [1] or [2] described, wherein, substituted amino acid is the amino acid of (7) and the amino acid of (12).

[8] according to the mutant enzyme [7] described, wherein, for the amino acid of (7), the amino acid after replacement is Histidine, and for the amino acid of (12), the amino acid after replacement is Serine, arginine, leucine or proline(Pro).

[9] according to the mutant enzyme [1] described, wherein, by the arbitrary aminoacid sequence in sequence number 7～21,59～61, formed.

[10] gene, the mutant enzyme in coding [1]～[9] described in any one.

[11] according to the gene [10] described, wherein, comprise the arbitrary base sequence in sequence number 22～36,62～64.

[12] recombinant DNA, comprises [10] or [11] described gene.

[13] microorganism, has [12] described recombinant DNA.

[14] methods for dextrose, is characterized in that, uses the mutant enzyme described in any one in [1]～[9], measures the glucose in sample.

[15] a glucose assays reagent, is characterized in that, comprise [1]～[9] in mutant enzyme described in any one.

[16] a glucose assays test kit, comprises [15] described glucose assays reagent.

[17] method, is characterized in that, uses the mutant enzyme described in any one in [1]～[9], reduces the glucose amount in mechanicals or its raw material.

[18] an enzyme agent, contain [1]～[9] in mutant enzyme described in any one.

[19] method of design for mutant enzyme, comprises the following steps (i) and (ii):

(i) in the aminoacid sequence of sudden change object enzyme, determine one or more the amino acid whose step being selected from following (1)～(13), said mutation object enzyme is from the glucose oxidase of microorganism or from the flavine-adenine-dinucleotide-dependent glucose dehydrogenase of microorganism:

(13) amino acid corresponding with 583 amino acids of the aminoacid sequence shown in sequence number 1;

(ii) the aminoacid sequence based on sudden change object enzyme, be structured in step in (i) definite aminoacid sequence by other aminoacid replacement and the step of aminoacid sequence.

[20] according to the method for design [19] described, wherein, sudden change object enzyme is the glucose oxidase from microorganism, step (i) in the substituted amino acid amino acid that is (3), (5) amino acid, the amino acid of (7) or the amino acid of (12) or be selected from the plural amino acid whose combination in them.

[21] according to the method for design [19] described, wherein, sudden change object enzyme is the glucose oxidase from microorganism, step (i) in substituted amino acid be (3) amino acid, the amino acid of (7) or the amino acid of (12) or be selected from the plural amino acid whose combination in them.

[22] method of design according to [19], wherein, sudden change object enzyme is the glucose oxidase from microorganism, step (i) in substituted amino acid be the amino acid of (7) and the amino acid of (12).

[23] according to the method for design described in any one in [20]～[22], wherein, from the glucose oxidase of microorganism, be the glucose oxidase of aspergillus niger (Aspergillus niger) or the rugged mould of Buddhist nun (Penicillium amagasakiense).

[24] according to the method for design [23] described, wherein, the aminoacid sequence of glucose oxidase is the aminoacid sequence of

sequence number

1 or 2.

[25] according to the method for design [19] described, wherein, sudden change object enzyme is the flavine-adenine-dinucleotide-dependent glucose dehydrogenase of Italian mould (Penicillium italicum), thin thorn mould (Penicillium lilacinoechinulatum), aspergillus oryzae (Aspergillus oryzae) or terreus (Aspergillus terreus).

[26] according to the method for design [25] described, wherein, the aminoacid sequence of flavine-adenine-dinucleotide-dependent glucose dehydrogenase is the arbitrary aminoacid sequence in sequence number 3～6.

[27] preparation method for mutant enzyme, comprise the following steps (I)～(III):

(I) step of the nucleic acid that the aminoacid sequence that preparation builds by the arbitrary aminoacid sequence in sequence number 7～21,59～61 or by the method for design described in any one in [19]～[26] is encoded;

(II) step that described nucleic acid is expressed, and

(III) step of recovery expression product.

Accompanying drawing explanation

Fig. 1 is the comparison with the aminoacid sequence of the GO from the rugged mould of Buddhist nun from the aminoacid sequence of the GO of aspergillus niger.With underscore represent the to suddenly change amino acid of object.With boldface letter, be illustrated in the amino acid of sudden change object and improve effective amino acid to GDH is active.Arrow is the amino acid in active centre." * " represents same (identical), and ": " represents that conservative property replaces (conservedsubstitutions), and ". " represents that semi-conservative property replaces (semi-conserved substitutions).

Fig. 2 is the comparison with the aminoacid sequence of the FAD-GDH from microorganism from the GO of microorganism.From epimere, start to represent successively the aminoacid sequence (the N-terminal side part of sequence number 4) of the aminoacid sequence (the N-terminal side part of sequence number 3) of Italian mould FAD-GDH, thin thorn mould FAD-GDH, the aminoacid sequence (the N-terminal side part of sequence number 5) of aspergillus oryzae FAD-GDH, the aminoacid sequence (the N-terminal side part of sequence number 6) of terreus FAD-GDH, the aminoacid sequence (the N-terminal side part of sequence number 1) of aspergillus niger GO.Be illustrated near the amino acid active centre in GO with underscore, different amino acid conservative between FAD-GDH (concomitant high) and between GO and FAD-GDH, and from N-terminal side successively append serial numbers.

Fig. 3 is the comparison (Fig. 1 continues) with the aminoacid sequence of the FAD-GDH from microorganism from the GO of microorganism.From epimere, start to represent successively the aminoacid sequence (the C-terminal side part of sequence number 4) of the aminoacid sequence (the C-terminal side part of sequence number 3) of Italian mould FAD-GDH, thin thorn mould FAD-GDH, the aminoacid sequence (the C-terminal side part of sequence number 5) of aspergillus oryzae FAD-GDH, the aminoacid sequence (the C-terminal side part of sequence number 6) of terreus FAD-GDH, the aminoacid sequence (the C-terminal side part of sequence number 1) of aspergillus niger GO.Be illustrated near the amino acid active centre in GO with underscore, different amino acid conservative between FAD-GDH (concomitant high) and between GO and FAD-GDH, from N-terminal side successively append serial numbers.Arrow is the amino acid in the active centre of GO.

Fig. 4 is the sequence (sequence number 37) comprising by the gene order of the aspergillus niger GO of pcr amplification.At 5' end, add HindIII site (frame line) and Kozak sequence (underscore), at 3' end side, add XhoI site (frame line).Should illustrate, by adding Kozak sequence, the 2nd amino acid is altered to Serine from glutamine.The gene order of aspergillus niger GO is as shown in sequence number 38.

Fig. 5 is the result of plate assay.The library (yeast saccharomyces cerevisiae (saccharomyces cerevisiae)) that making comprises the plasmid that imports sudden change, after copying the bacterium colony of growth, by plate assay detection GO activity and GDH activity in expression flat board.

Fig. 6 is the result that the activity during express liquid is cultivated is confirmed.For the transformant that can confirm positive bacterium colony (color development not in GO measures, color development in GDH measures) in mutant enzyme transformant, carry out liquid culture, relatively GO activity and GDH activity.Compare with sudden change (pYES-GO) not, represent the transformant that GDH activity value and GDH activity are simultaneously high with the ratio (GDH activity/GO activity) of GO activity with reticulate pattern.PYES-GO in table represents the transformant not suddenling change, pYES2 in table represents the transformant obtaining with inserting the plasmid conversion before gene, GO in table represents GO " Amano " 2(amano enzyme company), the FAD-GDH in table represents GDH " Amano " 8(amano enzyme company).

Fig. 7 is the chart that represents the result of confirming by the activity of liquid culture.With bar shaped, represent GOH activity and the GO activity of each mutant enzyme transformant, represent GDH activity/GO activity with broken line.PYES-GO represents the transformant not suddenling change.PYES-GO in chart represents the transformant not suddenling change, pYES2 in chart represents the transformant obtaining with inserting the plasmid conversion before gene, GO in chart represents GO " Amano " 2(amano enzyme company), the FAD-GDH in chart represents GDH " Amano " 8(amano enzyme company).

Fig. 8 represents to confirm that GDH/GO specific activity changes the table of the sudden change of mutant enzyme transformant greatly.Infer that the T353H of 5-1-5 results from mixing.Equally, infer that the D446S of 7-2-17 and the D446R of 7-2-42 also result from mixing.

Fig. 9 is the chart that represents the substrate specificity of saltant type GO.With the form of the reactive relative value to glucose, calculate the reactivity to each substrate.PYES-GO represents the transformant not suddenling change.

Figure 10 is the amino acid whose table of sudden change object being illustrated in each enzyme.Each enzyme has all been represented to (1) amino acid corresponding with 115 amino acids of the aminoacid sequence shown in sequence number 1, (2) amino acid corresponding with 131 amino acids of the aminoacid sequence shown in sequence number 1, (3) amino acid corresponding with 132 amino acids of the aminoacid sequence shown in sequence number 1, (4) amino acid corresponding with 193 amino acids of the aminoacid sequence shown in sequence number 1, (5) amino acid corresponding with 353 amino acids of the aminoacid sequence shown in sequence number 1, (6) amino acid corresponding with 436 amino acids of the aminoacid sequence shown in sequence number 1, (7) amino acid corresponding with 446 amino acids of the aminoacid sequence shown in sequence number 1, (8) amino acid corresponding with 472 amino acids of the aminoacid sequence shown in sequence number 1, (9) amino acid corresponding with 511 amino acids of the aminoacid sequence shown in sequence number 1, (10) amino acid corresponding with 535 amino acids of the aminoacid sequence shown in sequence number 1, (11) amino acid corresponding with 537 amino acids of the aminoacid sequence shown in sequence number 1, (12) amino acid corresponding with 582 amino acids of the aminoacid sequence shown in sequence number 1, (13) amino acid corresponding with 583 amino acids of the aminoacid sequence shown in sequence number 1.

Figure 11 is the active comparison of multiple mutation type GO.Take culture supernatant as sample, the GDH/GO specific activity of various transformants different sudden change combination is compared.Represent the sudden change that each mutant enzyme transformant has with zero.

Figure 12 is the specific activity with the mutant enzyme of the mutant enzyme transformant generation of the combination of effective sudden change.Represent the sudden change that each mutant enzyme transformant has with zero.

Figure 13 is the substrate specificity with the mutant enzyme of the mutant enzyme transformant generation of the combination of effective sudden change.The relatively reactivity to maltose, wood sugar, fructose, semi-lactosi, seminose and lactose.

Figure 14 is the active comparison of D446 and V582 multiple mutation enzyme.For the different various enzymes of amino acid after replacing, relatively GDH/GO specific activity.*: GO activity is below detectability.

Figure 15 is the substrate specificity of D446H and V582P multiple mutation enzyme.By the reactivity to maltose, wood sugar, fructose, semi-lactosi, seminose and lactose and FAD-GDH(GDH " Amano " 8(amano enzyme company)) compare.

Embodiment

For convenience of explanation, for the part for term of the present invention, be defined as follows.

(term)

Term " mutant enzyme " is for utilizing the disclosed method of this specification sheets that " basal enzyme " suddenlyd change and even modify the enzyme obtaining." mutant enzyme ", " mutant enzyme " and " modification type enzyme " commutative use.Basal enzyme typically is wild-type enzyme.But, do not hinder and as " basal enzyme ", be applicable to the present invention using being applied in manually-operated enzyme.Should illustrate, in this manual, " basal enzyme " is also known as " sudden change object enzyme " or " target enzyme ".

To make certain enzyme (being called for convenience of explanation A enzyme) and other enzyme (being called for convenience of explanation B enzyme) approximate, that is, so that the characteristic of more than one of A enzyme mode approaching with the corresponding characteristic of B enzyme modified, be called " A enzyme is carried out to B enzyme ".Here " characteristic " be exemplified as enzymic activity (for example glucose oxidase activity in the situation that A enzyme is glucose oxidase), substrate specificity, temperature profile (optimum temperuture, temperature stability etc.), pH characteristic (optimal pH, pH stability), coenzyme specificity, the reactivity with medium.

(glucose oxidase is suddenlyd change and enzyme)

The 1st aspect of the present invention relates to the enzyme (below also referred to as " sudden change GO ") that the glucose oxidase from microorganism (GO) is suddenlyd change and obtain.The present invention GO that suddenlys change has at the GO(sudden change object enzyme from microorganism) aminoacid sequence in be selected from one or more amino acid in following (1)～(13) by other aminoacid replacement and aminoacid sequence.

(1) amino acid corresponding with 115 amino acids of the aminoacid sequence shown in sequence number 1

(2) amino acid corresponding with 131 amino acids of the aminoacid sequence shown in sequence number 1

(3) amino acid corresponding with 132 amino acids of the aminoacid sequence shown in sequence number 1

(4) amino acid corresponding with 193 amino acids of the aminoacid sequence shown in sequence number 1

(5) amino acid corresponding with 353 amino acids of the aminoacid sequence shown in sequence number 1

(6) amino acid corresponding with 436 amino acids of the aminoacid sequence shown in sequence number 1

(7) amino acid corresponding with 446 amino acids of the aminoacid sequence shown in sequence number 1

(8) amino acid corresponding with 472 amino acids of the aminoacid sequence shown in sequence number 1

(9) amino acid corresponding with 511 amino acids of the aminoacid sequence shown in sequence number 1

(10) amino acid corresponding with 535 amino acids of the aminoacid sequence shown in sequence number 1

(11) amino acid corresponding with 537 amino acids of the aminoacid sequence shown in sequence number 1

(12) amino acid corresponding with 582 amino acids of the aminoacid sequence shown in sequence number 1

(13) amino acid corresponding with 583 amino acids of the aminoacid sequence shown in sequence number 1

As described later shown in embodiment, above-mentioned 115 amino acids, 131 amino acids, 132 amino acids, 193 amino acids, 353 amino acids, 436 amino acids, 446 amino acids, 472 amino acids, 511 amino acids, 535 amino acids, 537 amino acids, 582 amino acids and 583 amino acids are by the GO of aspergillus niger and multiple FAD-GDH are compared to the amino acid of finding, near the active centre in GO and have a GO characteristic.In the present invention, by the amino acid corresponding with these amino acid that makes to be considered to GO characteristic to play an important role, suddenly change, thereby realize improvement, the improvement of enzyme characteristic.

At this, the term " correspondence " while using for amino-acid residue in this manual refers between the protein being compared (enzyme) makes equal contribution to the performance of its function.For example, for reference amino acid sequence (, the aminoacid sequence of sequence number 1), by the aminoacid sequence of comparison other consider primary structure (aminoacid sequence) Homoeology in, can carry out optimum mode relatively while arranging (now can import as required room (gap) and make to compare optimization), the amino acid of the position corresponding with specific amino acids in reference amino acid sequence can be defined as to " corresponding amino acid ".Also can replace comparison each other of primary structure and utilize three-dimensional arrangement (three-D structure) comparison each other, or not only utilize primary structure comparison each other, but also utilize three-dimensional arrangement (three-D structure) comparison each other, determine " corresponding amino acid ".By utilizing three-dimensional arrangement information, can obtain the comparative result that reliability is high.In this case, can adopt the method for comparing when the atomic coordinate of the three-dimensional arrangement of plurality of enzymes is compared.The three-dimensional arrangement information of sudden change object enzyme for example can be obtained by Protein Data Bank (Protein Data Bank) (https://www.pdbj.org/index j.html).

Utilize the protein steric structure of X ray analysis of crystal structure determining method one example as follows.

(1) protein is carried out to crystallization.In order to determine three-dimensional arrangement, crystallization is indispensable, but in addition, as highly purified method of purification, high-density and the stable store method of protein, also has the availability in industry.In this case, preferably the protein that has substrate or its similar compound as ligand binding is carried out to crystallization.

(2), to the crystallization X-ray irradiation of making, collect diffraction data.Should illustrate, crystallization of protein suffers damage and diffraction can deteriorated situation be had much because of x-ray bombardment.In this case, crystallization is cooled to sharp to-173 ℃ of left and right, the cold test technology of collecting diffraction data under this state is universal gradually recently.Should illustrate, for final high de-agglomeration energy data of collecting for determining structure, the high synchrotron radiating light of brightness is utilized.

(3), when carrying out crystal structure analysis, except diffraction data, also need phase information.For target protein, when not knowing the crystalline structure of similar protein, can not determine structure with molecule method of substitution, must utilize heavy atom isomorphous substitution method to solve phase problem.Heavy atom isomorphous substitution method is following method: atoms metal large the atom sequence numbers such as mercury, platinum is imported in crystallization, utilize the large X ray scattering of atoms metal can obtain to the contribution of X ray diffracting data the method for phase information.The phase place determining can be by improving the electron density smoothing in the solvent region in crystallization.The water molecules in solvent region, because fluctuation does not observe greatly and almost electron density, therefore by the electron density that makes this region, is similar to 0, thereby can approaches real electron density, and even phase place improves.In addition,, while comprising different kinds of molecules in asymmetric unit, by the electron density of these molecules is averaged, thereby phase place obtains improvement by a larger margin.Make protein model meet the electron density map that uses the phase meter improving like this to calculate.This technique is used the QUANTA supervisor of MSI company (U.S.) to carry out on the basis of computer graphics.Then, use the X-PLOR supervisor of MSI company, carry out precise structure, complete structural analysis.For target protein, in the case of the similar crystalline structure of protein be known, use the atomic coordinate of known protein, utilize molecule method of substitution to determine.Molecule replacement is carried out with precise structure available programs CNS SOLVE ver.11 etc.

Sudden change object enzyme i.e. being exemplified as from the GO of aspergillus niger and from the GO of the rugged mould of Buddhist nun from the GO of microorganism.The aminoacid sequence of the GO from aspergillus niger registering in public database and from the aminoacid sequence of the GO of the rugged mould of Buddhist nun respectively as shown in sequence number 1 and sequence number 2.In addition, the comparison of this two seed amino acids sequence more as shown in Figure 1.

At the GO from aspergillus niger of the aminoacid sequence to there is sequence number 1 during as sudden change object enzyme, the amino acid of above-mentioned (1) is 115 amino acids of sequence number 1, the amino acid of above-mentioned (2) is 131 amino acids of sequence number 1, the amino acid of above-mentioned (3) is 132 amino acids of sequence number 1, the amino acid of above-mentioned (4) is 193 amino acids of sequence number 1, the amino acid of above-mentioned (5) is 353 amino acids of sequence number 1, the amino acid of above-mentioned (6) is 436 amino acids of sequence number 1, the amino acid of above-mentioned (7) is 446 amino acids of sequence number 1, the amino acid of above-mentioned (8) is 472 amino acids of sequence number 1, the amino acid of above-mentioned (9) is 511 amino acids of sequence number 1, the amino acid of above-mentioned (10) is 535 amino acids of sequence number 1, the amino acid of above-mentioned (11) is 537 amino acids of sequence number 1, the amino acid of above-mentioned (12) is 582 amino acids of sequence number 1, the amino acid of above-mentioned (13) is 583 amino acids of sequence number 1.

On the other hand, at the GO from the rugged mould of Buddhist nun of the aminoacid sequence to there is sequence number 2 during as sudden change object enzyme, the amino acid of above-mentioned (1) is 115 amino acids of sequence number 2, the amino acid of above-mentioned (2) is 131 amino acids of sequence number 2, the amino acid of above-mentioned (3) is 132 amino acids of sequence number 2, the amino acid of above-mentioned (4) is 193 amino acids of sequence number 2, the amino acid of above-mentioned (5) is 353 amino acids of sequence number 2, the amino acid of above-mentioned (6) is 436 amino acids of sequence number 2, the amino acid of above-mentioned (7) is 446 amino acids of sequence number 2, the amino acid of above-mentioned (8) is 472 amino acids of sequence number 2, the amino acid of above-mentioned (9) is 511 amino acids of sequence number 2, the amino acid of above-mentioned (10) is 535 amino acids of sequence number 2, the amino acid of above-mentioned (11) is 537 amino acids of sequence number 2, the amino acid of above-mentioned (12) is 582 amino acids of sequence number 2, the amino acid of above-mentioned (13) is 583 amino acids of sequence number 2.

Substituted amino acid is preferably the amino acid of (3), amino acid, the amino acid of (7) or the amino acid of (12) of (5).These shown in embodiment, are the amino acid that the raising of GDH activity is effectively confirmed as described later.In at least one substituted sudden change GO in these amino acid, with sudden change before enzyme compared with can bring into play high GDH activity.(3) enzyme of the concrete example of the substituted sudden change of amino acid GO for being formed by the aminoacid sequence of sequence number 7.Equally, (5) enzyme of the concrete example of the substituted sudden change of amino acid GO for being formed by the aminoacid sequence of sequence number 8, (7) enzyme of the concrete example of the substituted sudden change of amino acid GO for being formed by the aminoacid sequence of sequence number 9, the enzyme of the concrete example of the substituted sudden change of the amino acid GO of (12) for being formed by the aminoacid sequence of sequence number 10.These sudden changes GO all demonstrates high GDH activity with the enzyme before sudden change compared with the GO of aspergillus niger.

Amino acid whose kind after replacement is not particularly limited, but preferably not belong to the mode of so-called " conservative amino acid replacement ", selects the amino acid after replacing.Here " conservative amino acid replacement " refers to certain amino-acid residue is replaced to the amino-acid residue with ejusdem generis side chain.Amino-acid residue is divided into basic side chain (for example Methionin according to its side chain, arginine, Histidine), acid side-chain (for example aspartic acid, L-glutamic acid), non-charged polar side chain (for example glycine, asparagine, glutamine, Serine, Threonine, tyrosine, halfcystine), non-polar sidechain (for example L-Ala, α-amino-isovaleric acid, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met), tryptophane), β branched building block (for example Threonine, α-amino-isovaleric acid, Isoleucine), aromatic series side chain (for example tyrosine, phenylalanine, tryptophane, Histidine) and so on some families.Conservative amino acid replaces the replacement between the amino-acid residue typically being in same gang.

If enumerate the amino acid whose example after replacement, for the amino acid of (3), be L-Ala, be L-Ala for the amino acid of (5), is Histidine for the amino acid of (7), for the amino acid of (12), be Serine, arginine, leucine and proline(Pro).

In the amino acid of above-mentioned (1)～(13), two or more amino acid can be replaced.List as follows the example of substituted amino acid whose combination.

(3) with the combination of (5)

(3) with the combination of (7)

(3) with the combination of (12)

(5) with the combination of (7)

(5) with the combination of (12)

(7) with the combination of (12)

(3), the combination of (5) and (7)

(3), the combination of (5) and (12)

(3), the combination of (7) and (12)

(5), the combination of (7) and (12)

(3), the combination of (5), (7) and (12)

The example of the aminoacid sequence of the mutant enzyme of applying above combination and obtain is as shown in sequence number 11～21.These sequences are the aminoacid sequence of the sudden change GO that obtains for the GO application aforesaid combination of aspergillus niger.The corresponding relation of the combination of sequence number and sudden change is as described below.

Sequence number 11:(3) with the combination of (5)

Sequence number 12:(3) with the combination of (7)

Sequence number 13:(3) with the combination of (12)

Sequence number 14:(5) with the combination of (7)

Sequence number 15:(5) with the combination of (12)

Sequence number 16:(7) with the combination of (12)

Sequence number 17:(3), the combination of (5) and (7)

Sequence number 18:(3), the combination of (5) and (12)

Sequence number 19:(3), the combination of (7) and (12)

Sequence number 20:(5), the combination of (7) and (12)

Sequence number 21:(3), the combination of (5), (7) and (12)

Sequence number 59:(7) with the combination of (12)

Sequence number 60:(7) with the combination of (12)

Sequence number 61:(7) with the combination of (12)

If according to the experimental result representing in aftermentioned embodiment (confirm sudden change combination effect), in above combination, the preferably combination of (3) and (12), the combination of (7) and (12), and the combination of (3), (7) and (12).The combination (concrete example of the aminoacid sequence of the mutant enzyme of this combination is

sequence number

16,59～61 as mentioned above) of particularly preferred being combined as (7) and (12).Amino acid after replacement is preferred group propylhomoserin for (7), preferred Serine for (12) (sequence number 16), arginine (sequence number 59), leucine (sequence number 60) or proline(Pro) (sequence number 61).For (12), the amino acid after particularly preferably replacing is proline(Pro).

But, generally speaking, when the part of aminoacid sequence that makes certain protein is suddenlyd change, sometimes the protein after sudden change have with sudden change before the equal function of protein.That is, the sudden change of aminoacid sequence does not sometimes bring substantial impact for the function of protein, and the function of protein is maintained before and after sudden change.If consider this technology general knowledge, with above-mentioned by being selected from (1)～(13) in one or more amino acid by other aminoacid replacement and the sudden change GO that aminoacid sequence formed while comparing, although the slightly different not confirmed of aminoacid sequence (wherein, the position that not the coexisting of aminoacid sequence implemented beyond the position of above-mentioned aminoacid replacement occurs), but in characteristic, the enzyme of substantial difference not confirmed can be considered to the enzyme same with said mutation GO essence.Here " aminoacid sequence slightly different " typically refer to and utilize 1～several (being above limited to for example 3,5,7,10) the amino acid whose disappearance, the replacement that form aminoacid sequence, or 1～several (being above limited to for example 3,5,7,10) amino acid whose interpolation, insertion or these combinations and in aminoacid sequence, produce sudden change (variation).The aminoacid sequence of " enzyme that essence is same " is preferably more than 90% with the identity (%) of the aminoacid sequence of the said mutation GO as benchmark, more preferably more than 95%, more preferably more than 98%, most preferably is more than 99%.Should illustrate, the difference of aminoacid sequence can produce in multiple positions." aminoacid sequence slightly different " preferably replaced and produced by conservative amino acid.

(nucleic acid of encoding mutant GO etc.)

The 2nd aspect of the present invention provides the relevant nucleic acid of GO that suddenlys change to the present invention.That is, provide and can serve as the nucleic acid using for the probe of the nucleic acid of the gene of identifier number sudden change GO, encoding mutant GO; The nucleic acid that can use as the primer of the nucleic acid amplification for making encoding mutant GO or sudden change etc.

The gene of encoding mutant GO is typically for the preparation of the GO that suddenlys change.According to the preparation method of the genetic engineering of the gene of use encoding mutant GO, can obtain the more sudden change GO of homogeneous state.In addition, the method also can be described as preferred method when preparing a large amount of sudden change GO.In addition the purposes of the gene of the encoding mutant GO preparation of GO that is not limited to suddenly change.For example, the experiment instrument as the explanation take sudden change GO mechanism of action etc. as object, or as the instrument that is used for the further mutant that designs or make enzyme, also can utilize this nucleic acid.

In this manual, so-called " gene of encoding mutant GO " refers to the nucleic acid that obtains this sudden change GO when making this genetic expression, certainly comprise the nucleic acid with the base sequence corresponding with the aminoacid sequence of this sudden change GO, be also included in and in this nucleic acid, be attached with the nucleic acid that sequence of encoding amino acid sequence does not form.In addition also consider, the degeneracy of codon.

The example of the sequence of the gene of encoding mutant GO is as shown in sequence number 22～36,62～64.These sequences are to be coded in the gene of having implemented the sudden change GO that specific aminoacid replacement obtains in the GO of aspergillus niger.Aminoacid replacement in each sequence is as described below.

Sequence number 22:T132A

Sequence number 23:T353A

Sequence number 24:D446H

Sequence number 25:V582S

Sequence number 26:T132A and T353A

Sequence number 27:T132A and D446H

Sequence number 28:T132A and V582S

Sequence number 29:T353A and D446H

Sequence number 30:T353A and V582S

Sequence number 31:D446H and V582S

Sequence number 32:T132A, T353A and D446H

Sequence number 33:T132A, T353A and V582S

Sequence number 34:T132A, D446H and V582S

Sequence number 35:T353A, D446H and V582S

Sequence number 36:T132A, T353A, D446H and V582S

Sequence number 62:D446H and V582R

Sequence number 63:D446H and V582L

Sequence number 64:D446H and V582P

Nucleic acid of the present invention can be with reference to this specification sheets or the disclosed sequence information of appended sequence table, by using the genetic engineering method, molecular biology method, biochemical method etc. of standard, is prepared into separate stage.

In alternate manner of the present invention, a kind of nucleic acid is provided, it is compared with the base sequence of the gene of code book invention sudden change GO time, although the function of the protein of its coding is identical, a part of base sequence difference is (below, also referred to as " homologous nucleic acid ".In addition, by regulation homologous nucleic acid base sequence also referred to as " homology base sequence ").As the example of homologous nucleic acid, can enumerate the following protein DNA of following coding, the base sequence of replacement, disappearance, insertion, interpolation or inversion that described protein comprises 1 or multiple bases by the base sequence of the nucleic acid with code book invention sudden change GO by benchmark is formed, and there is the sudden change distinctive enzymic activity of GO (that is, GDH activity).The replacement of base, disappearance etc. can produce at multiple positions.Here " multiple " are different and different according to the position of the amino-acid residue in the three-dimensional arrangement of the protein of this nucleic acid encoding, kind, for example, be 2～40 bases, are preferably 2～20 bases, more preferably 2～10 bases.

Homologous nucleic acid as above for example can obtain as follows: restriction enzyme processing; Utilize the processing of exonuclease, DNA ligase etc.; Utilize positional mutation introductory technique (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York), random mutation introductory technique (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York) sudden change importing etc.In addition, by other methods such as uviolizings, also can obtain homologous nucleic acid.

Alternate manner of the present invention relates to a kind of nucleic acid, and it has the base sequence with respect to the base sequence complementation of the gene of code book invention sudden change GO.Further alternate manner of the present invention provides a kind of nucleic acid, and it has with respect to base sequence or the base sequence complementary with it of the gene of code book invention sudden change GO and is at least about 60%, 70%, 80%, 90%, 95%, 99%, 99.9% same base sequence.

Further alternate manner of the present invention relates to a kind of nucleic acid, and it has with respect to the base sequence of the gene of code book invention sudden change GO or the base sequence of hybridizing under rigorous type condition with the base sequence of its homology base sequence complementation.Here " rigorous type condition " refers to the condition that forms so-called specific hybrid and do not form non-specific hybridization.Rigorous type condition is like this well known to a person skilled in the art, for example can be with reference to Molecular Cloning(Third Edition, Cold Spring Harbor Laboratory Press, New York), Current protocols in molecular biology(edited by Frederick M.Ausubel et al., 1987) set.As rigorous type condition, for example can enumerate with hybridization solution (50% formaldehyde, 10 × SSC(0.15MNaCl, 15mM Trisodium Citrate, pH 7.0), 5 × Denhardt solution, 1%SDS, 10% T 500, the denatured salmon sperm dna of 10 μ g/ml, 50mM phosphoric acid buffer (pH7.5)), at approximately 42 ℃～approximately 50 ℃, carry out incubation, the condition of then using 0.1 × SSC, 0.1%SDS to clean at approximately 65 ℃～approximately 70 ℃.As further preferred rigorous type condition, for example can enumerate, as hybridization solution, use 50% formaldehyde, 5 × SSC(0.15M NaCl, 15mM Trisodium Citrate, pH 7.0), 1 × Denhardt solution, 1%SDS, 10% T 500, the denatured salmon sperm dna of 10 μ g/ml, 50mM phosphoric acid buffer (pH7.5)) condition.

Further alternate manner of the present invention provides a kind of nucleic acid (nucleic acid fragment), and it has the base sequence of gene or a part for the base sequence complementary with it of code book invention sudden change GO.This nucleic acid fragment can be used for the nucleic acid etc. of base sequence of gene with code book invention sudden change GO to detect, differentiate and/or amplification etc.Nucleic acid fragment is design as follows for example: continuous nucleotide segment (for example approximately 10～approximately 100 base length in the base sequence of the gene of code book invention sudden change GO, preferably approximately 20～approximately 100 base length, more preferably from about 30～approximately 100 base length) at least comprise hybridization portion.When as probe, labelable nucleic acid fragment.In mark, for example can use fluorescent substance, enzyme, radio isotope.

Further alternate manner of the present invention relates to the recombinant DNA that comprises gene of the present invention (gene of encoding mutant GO).Recombinant DNA of the present invention for example provides with the form of carrier.Term " carrier " refers to and the nucleic acid inserting wherein can be transported to the core acidic molecular in target in this manual.

According to application target (clone, protein expression), consider in addition the kind of host cell and select suitable carrier.The carrier that is host as colibacillus, can illustrate M13 phage or its modification body, lambda particles phage or its modification body, pBR322 or its modification body (pB325, pAT153, pUC8 etc.) etc., as the carrier take yeast as host, can illustrate pYepSec1, pMFa, pYES2 etc., as the carrier take insect cell as host, pAc, pVL etc. can be illustrated, as the carrier take cells of mamma animals as host, pCDM8, pMT2PC etc. can be illustrated.

Carrier of the present invention is preferably expression vector." expression vector " refers to and the nucleic acid inserting wherein can be imported in target cell (host cell), and can make it the carrier of expressing in this cell.Expression vector conventionally comprises the promoter sequence essential to the expression of inserted nucleic acid, promotes the enhancer sequence of expression etc.Also can use the expression vector that comprises selective marker.Using during this expression vector, can utilize selective marker and confirm have or not (and degree) that expression vector imports.

The recombinant DNA technology that nucleic acid of the present invention can be used standard to insertion (if desired) of the insertion of carrier, the insertion of selectable marker gene (if desired), promotor etc. (for example, can be with reference to Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, known method New York, that use restriction enzyme and DNA ligase) carry out.

As host cell, from the viewpoint of processing ease, preferably use the microorganisms such as colibacillus (Escherichiacoli), budding yeast (Saccharomyces cerevisiae), but as long as recombinant DNA is reproducible and the effable host cell of gene of sudden change GO can utilize.As the example of colibacillus, when utilizing T7 to be promotor, can enumerate colibacillus BL21(DE3) pLysS can enumerate colibacillus JM109 when being not such.In addition,, as the example of budding yeast, can enumerate budding yeast SHY2, budding yeast AH22 or budding yeast INVSc1(Invitrogen company).

Other side of the present invention relates to the microorganism (that is, transformant) with recombinant DNA of the present invention.Microorganism of the present invention can utilize uses the transfection of the invention described above carrier and even conversion and obtains.For example, can utilize Calcium Chloride Method (Journal of Molecular Biology(J.Mol.Biol.), the 53rd volume, the 159th page (1970)), Hanahan method (Journal of Molecular Biology, the 166th volume, the 557th page (1983)), SEM method (gene (Gene), the 96th volume, the 23rd page (1990)), method (the Proceedings of the National Academy of Sciences of the United States ofAmerica of Chung etc., the 86th volume, the 2172nd page (1989)), calcium phosphate is Shen Jiangfa altogether, electroporation (Potter, H.et al., Proc.Natl.Acad.Sci.U.S.A.81, 7161-7165(1984)), lipofection (Felgner, P.L.et al., Proc.Natl.Acad.Sci.U.S.A.84, the enforcement such as 7413-7417(1984)).Should illustrate, microorganism of the present invention can be used for producing the preparation method hurdle of sudden change GO(of the present invention with reference to aftermentioned mutant enzyme).

(purposes of sudden change GO)

The 3rd mode of the present invention relates to the purposes of the GO that suddenlys change.In which, first provide the methods for dextrose that uses sudden change GO.In methods for dextrose of the present invention, utilize by the redox reaction due to this enzyme, measure the glucose amount in sample.The inventive example is as mensuration of the glucose concn of the mensuration for blood glucose value, food (food flavouring, beverage etc.) etc.In addition, for example, for example, in the manufacturing process of leavened food (vinegar) or fermented drink (beer, wine), in order to study fermentation degree, also can utilize the present invention.

The present invention also provides the glucose assays that comprises this enzyme reagent.This reagent is for the methods for dextrose of the invention described above.

The present invention further provides the test kit (glucose assays test kit) for implementing methods for dextrose of the present invention.Test kit of the present invention, except the glucose assays that comprises this enzyme is with reagent, also comprises reaction reagent, damping fluid, glucose reference liquid etc. as any key element.In addition, in glucose assays test kit of the present invention, conventionally have working instructions.

The present invention, as further purposes, provides by making sudden change GO of the present invention to the mechanicals (food such as various processed foods, snack categories, refreshment drink water, alcoholic beverage, dietary supplement; Cosmetic etc.) or its raw material etc. act on and make method that glucose content reduces and the enzyme agent for this purposes.For example, when GO that the present invention is suddenlyd change is applied to food, can suppress Maillard reaction by reducing glucose content.Enzyme agent of the present invention, except effective constituent (sudden change GO), also can contain vehicle, buffer reagent, suspensoid, stablizer, preservatives, sanitas, physiological saline etc.As vehicle, can use starch, dextrin, maltose, trehalose, lactose, D-Glucose, sorbyl alcohol, D-mannital, white sugar, glycerine etc.As buffer reagent, can use phosphoric acid salt, Citrate trianion, acetate etc.As stablizer, can use propylene glycol, xitix etc.As preservatives, can use phenol, benzalkonium chloride, benzylalcohol, butylene-chlorohydrin, methyl hydroxybenzoate etc.As sanitas, can use ethanol, benzalkonium chloride, P-hydroxybenzoic acid, butylene-chlorohydrin etc.

(method of design of mutant enzyme)

Another aspect of the present invention relates to the method for design of mutant enzyme.In method of design of the present invention, implement following steps (i) and (ii).

Step is (i): in the aminoacid sequence of the object enzyme that suddenlys change at the glucose oxidase from microorganism (from the GO of microorganism) or from the flavine-adenine-dinucleotide-dependent glucose dehydrogenase (from the FDA-GDH of microorganism) of microorganism, determine the amino acid that is selected from following one or more.

Above-mentioned replacement object amino acid (1)～(13) utilize and find from the GO of microorganism and the comparison of the multiple FAD-GDH from microorganism.Expectation changes the characteristic of enzyme by replacing these amino acid.If enumerate the example of the characteristic that can change, be GO activity, GDH activity, substrate specificity, temperature profile (optimum temperuture, temperature stability etc.), pH characteristic (optimal pH, pH stability), coenzyme specificity, the reactivity with medium.

Sudden change object enzyme in method of design of the present invention is from the GO of microorganism or from the FAD-GDH of microorganism.Sudden change object enzyme typically is wild-type enzyme (enzyme of natural discovery).But the enzyme that does not hinder to have implemented certain sudden change and even modification is sudden change object enzyme.From the GO that is exemplified as aspergillus niger of GO and the GO of the rugged mould of Buddhist nun of microorganism, from the FAD-GDH that is exemplified as Italian mould, FAD-GDH, the FAD-GDH of aspergillus oryzae and the FAD-GDH of terreus of thin thorn mould of the FAD-GDH of microorganism.As the aminoacid sequence at this illustrative enzyme, the sequence of registering in public database is as follows.Should illustrate, in a preferred mode, the enzyme being formed using the arbitrary amino acid sequence by is wherein as sudden change object enzyme.

The GO of aspergillus niger (Aspergillus niger): the aminoacid sequence of sequence number 1

The GO of the rugged mould of Buddhist nun (Penicillium amagasakiense): the aminoacid sequence of sequence number 2

The FAD-GDH of Italy's mould (Penicillium italicum): the aminoacid sequence of sequence number 3

The FAD-GDH of thin thorn mould (Penicillium lilacinoechinulatum): the aminoacid sequence of sequence number 4

The FAD-GDH of aspergillus oryzae (Aspergillus oryzae): the aminoacid sequence of sequence number 5

The FAD-GDH of terreus (Aspergillus terreus): the aminoacid sequence of sequence number 6

Should illustrate, for above illustrative each enzyme, the amino acid whose amino acid that belongs to above-mentioned (1)～(13) be gathered in the table that is shown in Figure 10.

In the present invention step (i) after, carry out following steps (ii).

Step is (ii): based on the aminoacid sequence of sudden change object enzyme, be formed in step in (i) definite aminoacid sequence by the aminoacid sequence of other aminoacid replacement.

Amino acid whose kind after replacement is not particularly limited.Therefore, can replace for conservative amino acid, can be also non-conservation aminoacid replacement.Here " conservative amino acid replacement " refers to certain amino-acid residue is replaced to the amino-acid residue with ejusdem generis side chain.Amino-acid residue can be divided into base side chain (for example Methionin according to its side chain, arginine, Histidine), acid side-chain (for example aspartic acid, L-glutamic acid), non-charged polar side chain (for example asparagine, glutamine, Serine, Threonine, tyrosine, halfcystine), non-polar sidechain (for example glycine, L-Ala, α-amino-isovaleric acid, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met), tryptophane), β branched building block (for example Threonine, α-amino-isovaleric acid, Isoleucine), aromatic series side chain (for example tyrosine, phenylalanine, tryptophane) and so on some families.Conservative amino acid replaces the replacement between the amino-acid residue being preferably in same gang.

(preparation method of mutant enzyme)

Another way of the present invention relates to the preparation method of mutant enzyme.In a mutant enzyme preparation method's of the present invention mode, the sudden change GO genetic engineering method that the successes such as the present inventor are obtained is prepared.The in the situation that of which, prepare the nucleic acid (step (I)) of the arbitrary amino acid sequence of encoding sequence numbers 7～10.At this, " nucleic acid of the specific aminoacid sequence of encoding " is at the nucleic acid that makes it can obtain the in the situation that of expression the polypeptide with this aminoacid sequence, certainly can be the nucleic acid being formed by the base sequence corresponding with this aminoacid sequence, also can in such nucleic acid, add unnecessary sequence (can be the sequence of encoding amino acid sequence, can be also not the sequence of encoding amino acid sequence).In addition also consider, the degeneracy of codon." nucleic acid of the arbitrary amino acid sequence of encoding sequence numbers 7～10 " is with reference to this specification sheets or the disclosed sequence information of appended sequence table, by using the genetic engineering method, molecular biology method, biochemical method etc. of standard, can be prepared into separate stage.At this, the aminoacid sequence of sequence number 7～10 be to the aminoacid sequence of the GO from aspergillus niger implement sudden change and.Therefore, the gene (sequence number 38) for coding from the GO of aspergillus niger, by applying necessary sudden change, also can obtain the nucleic acid (gene) of the arbitrary amino acid sequence in encoding sequence numbers 7～10.The method replacing for location specific base sequence this technical field known have a lot (for example, with reference to MolecularCloning, Third Edition, Cold Spring Harbor Laboratory Press, New York), can be by the appropriate method of choice for use wherein.As location specific sudden change introductory technique, can adopt location specific amino acid saturation mutation method.Location specific amino acid saturation mutation method is the three-dimensional arrangement based on albumen, infer the relative position of desired function, import " half reasoning half random (Semi-rational, semi-random) " method (J.Mol.Biol.331,585-592(2003) of amino acid saturation mutation).For example, can use Quick change(Stratagene company) etc. test kit, Overlap extention PCR(Nucleic Acid Res.16,7351-7367(1988)) and import location specific amino acid saturation mutation.Archaeal dna polymerase for PCR can be used Taq polysaccharase etc.But, preferably use KOD-PLUS-(Japan to spin society), Pfu turbo(Stratagene company) etc. the high archaeal dna polymerase of precision.

In other mode of the present invention, based on utilizing the aminoacid sequence of method of design design of the present invention, prepare mutant enzyme.The in the situation that of which, in step (I), prepare the nucleic acid that the aminoacid sequence that utilizes method of design of the present invention and build is encoded.For example, based on the aminoacid sequence that utilizes method of design of the present invention to build, for the gene of encoding mutant object enzyme, apply necessary sudden change (that is, being the amino acid whose replacement of the specific position in protein at expression product), obtain the nucleic acid (gene) of encoding mutant enzyme.

Step (I) afterwards, makes the nucleic acid of preparing express (step (II)).For example, first, prepare inserted above-mentioned nucleic acid and expression vector, use this expression vector transformed host cell." expression vector " refers to and the nucleic acid inserting wherein can be imported in target cell (host cell), and can in this cell, make it the carrier of expressing.Expression vector comprises the promoter sequence of expression necessity of the nucleic acid to inserting, the enhancer sequence that promotion is expressed etc. conventionally.Also can use the expression vector that comprises selective marker.Using during this expression vector, can utilize selective marker and confirm have or not (and degree) that expression vector imports.

Then producing expression product, be, to cultivate transformant under the condition of mutant enzyme.The cultivation of transformant is according to ordinary method.As the carbon source for substratum, as long as assimilable carbon compound, for example, use glucose, sucrose, lactose, maltose, molasses, pyruvic acid etc.In addition,, as nitrogenous source, as long as available nitrogen compound, for example, use peptone, meat extract, yeast extract, casein hydrolysate, soybean residue alkaline extraction thing etc.In addition, use as required the salt of phosphoric acid salt, carbonate, vitriol, magnesium, calcium, potassium, iron, manganese, zinc etc.; Specific amino acid; Specific VITAMIN etc.

On the other hand, culture temperature can be set in the scope of 30 ℃～40 ℃ (preferably 37 ℃ near).Incubation time can be considered the growth characteristics of the transformant of Object of Development, the generation characteristic of mutant enzyme etc. and set.The pH of substratum is in the scope internal modulation of growth transformant and generation enzyme.Preferably the pH of substratum is made as near 6.0～9.0 left and right (preferably pH7.0).

Then, reclaim expression product (mutant enzyme) (step (III)).The nutrient solution that comprises thalline that can be after cultivating is directly as enzyme solution utilization, or through after removing of concentrated, impurity etc. as enzyme solution utilization, but generally speaking, expression product is temporarily reclaimed from nutrient solution or thalline.If expression product is secretary protein, from nutrient solution, reclaim, in addition, can in thalline, reclaim.When reclaiming from nutrient solution, for example culture supernatant is filtered, centrifugal treating and remove insolubles, then by concentrating under reduced pressure, membrane concentration, utilize ammonium sulfate, saltouing of sodium sulfate, utilize methyl alcohol, the step-by-step precipitation method of ethanol or acetone etc., dialysis, heat treated, iso-electric point processing, gel-filtration, adsorption chromatography, ion exchange chromatography, the various chromatographys such as affinity chromatography (for example, utilize the gel-filtration of dextrane gel (GE Healthcare Bioscience company) etc., DEAE agarose gel cl-6b (GE Healthcare Bioscience company), octyl sepharose gel C L-6B(GE Healthcare Bioscience company), CM agarose gel cl-6b (GE Healthcare Bioscience company)) etc. combination and separating, purifying, thereby obtain the purifying product of mutant enzyme.On the other hand, when reclaiming in thalline, by nutrient solution is filtered, centrifugal treating etc., gather thalline, then by the mechanical means such as pressure treatment, ultrasonication for thalline or after utilizing the enzyme process of lysosome etc. to destroy, with above-mentioned same separation, the purifying of carrying out, thereby can obtain the purifying product of mutant enzyme.

Also the purifying enzyme obtaining as mentioned above for example can be utilized to the powdered such as lyophilize, vacuum-drying or spraying are dried and be provided.Now, can make in advance purifying enzyme predissolve in the damping fluid of phosphoric acid buffer, trolamine damping fluid, tris hydrochloride buffer, GOOD.Can preferably use phosphoric acid buffer, trolamine damping fluid.Should illustrate, at this, as the damping fluid of GOOD, can enumerate PIPES, MES or MOPS.

Conventionally, utilize as mentioned above suitable host-vector to be and carry out the recovery of the expression～expression product (mutant enzyme) of gene, but also can utilize cell free translation system.At this, " cell free translation system (cell-free transcription system, cell-free transcription/translation system) " refers to and do not use viable cell, but use from (or with genetic engineering method obtain) rrna of viable cell, the transcription and translation factor etc., from external mRNA, the protein that synthesizes its coding of template nucleic acid (DNA, mRNA).In cell free translation system, generally use the cell extract that cytoclasis liquid is carried out to purifying as required and obtain.In cell extract, generally comprise the various enzymes such as the various factors, tRNA such as rrna to protein synthesis necessity, initiation factor.When carrying out protein synthetic, in this cell extract, add other material of the synthetic necessity to protein such as each seed amino acid, ATP, GTP homenergic source, creatine phosphate.Certainly, when synthetic protein, can supplement as required rrna, the various factor and/or the various enzymes etc. prepared separately.

Also reported the exploitation (Shimizu, Y.et al.:Nature Biotech., 19,751-755,2001) of the transcribe/translation system that reconstructs the each molecule (factor) to protein synthesis necessity.In this synthetic system; by the elongation factor of 3 kinds of initiation factors of the protein synthesis system of formation bacterium, 3 kinds, 4 kinds of factors that participate in termination, the 20 seed amino acid acyl group tRNA synthetic enzyme that each amino acid is combined with tRNA; and the gene of 31 kinds of factors that consist of methionyl transfer RNA formylation transferring enzyme increases from colibacillus genome; use these, the external protein synthesis system that reconstructs.Can utilize in the present invention such synthesis system that reconstructs.

Term " cell-free transcription/translation system " can be used with cell-free protein synthesis system, external translating system or in-vitro transcription/translation system exchange.In translation system, RNA is used as template and synthetic protein in vitro.As template ribonucleic acid, use total RNA, mRNA, in-vitro transcription product etc.In other in-vitro transcription/translation system, DNA is used as template.Template DNA should comprise rrna calmodulin binding domain CaM, preferably comprises in addition appropriate terminator sequence.Should illustrate, in transcribe in vitro/translation system, the mode of carrying out continuously with responsive transcription and translation reaction, sets the condition that the necessary factor of each reaction is added.

Embodiment

Creating under the such target of the high GDH of practicality, finding out the novel method of replacement method (method that existing GDH is modified, the method centered by screening) in the past.First be conceived to not have the glucose oxidase (GO) of the distinctive problem of FAD-GDH (reactivity for wood sugar is higher, and optimum temperuture is high).The homology of the aminoacid sequence of known GO and FAD-GDH is higher.Adopt following novel method: pay attention to this homology, to GO, give GDH activity, that is, utilize to modify GO carry out GDHization.

1.GO and FAD-GDH compare comparison

From the GO from aspergillus niger and the comparison of comparing from the FAD-GDH of the known aspergillus oryzae of current aminoacid sequence, terreus, Italian mould, thin thorn mould, and the three-dimensional arrangement of the clear and definite GO from aspergillus niger of three-dimensional arrangement, in near the amino acid active centre in GO, conservative between FAD-GDH (concomitant high), but between GO and FAD-GDH, retrieve different amino acid (Fig. 2,3).Should illustrate, comparison relatively in, be provided with the Laboratory at ClustalW2(EMBL(European Molecular Biology)-EBI(European Bioinformatics Institute) homepage in special site.Use https://www.ebi.ac.uk/Tools/clustalw2/index.html).

To utilize the amino acid of 13 (L115, G131, T132, V193, T353, F436, D446, Y472, I511, P535, Y537, V582, M583) that retrieval is determined to import object as suddenling change.

Obtaining, suddenling change of 2.GO gene imports and plate assay

For GO gene, the report that the past does not express in colibacillus, therefore determines the company at pYES2(Invitrogen) HindIII-XhoII part in insert GO gene, take yeast saccharomyces cerevisiae (S.cerevisiae) as host, make it to express.

From Gen Elute Plant Genomic DNA kit(Sigma company for aspergillus niger GO-1 bacterium (amano enzyme company has)) extract genomic dna, utilize PCR to obtain GO gene.Below, represent the condition of PCR.

(composition of reaction solution)

10 × LA damping fluid (Takara-bio Co., Ltd.), 5 μ L

2.5mM dNTPs(Takara-bio Co., Ltd.) 8 μ L

25mM MgCl ₂(Takara-bio Co., Ltd.) 5 μ L

Forward primer (50 μ M) 1 μ L

Reverse primer (50 μ M) 1 μ L

Template 1 μ L

LA Taq(Takara-bio Co., Ltd.) 0.5 μ L

stH ₂O 28.5μL

(primer sequence)

Forward primer: GATCAGAAGCTTAAAAAAATGTCTACTCTCCTTGTGAGCTCG(sequence number 39)

Reverse primer: GATCAGCTCGAGTCACTGCATGGAAGCATAATC(sequence number 40)

(reaction conditions)

In 94 ℃ reaction 2 minutes after, by 94 ℃ reaction 30 seconds, in 52 ℃ reaction 30 seconds, in 72 ℃ reaction 2 minutes reaction cycle repeat 35 times, then in 72 ℃ reaction 7 minutes, finally in 4 ℃ of placements.

Amplified production after PCR is inserted to pYES2, make pYES-GO-K-P-2 plasmid, confirmed the sequence (Fig. 4) of inset.Owing to not pinpointing the problems in sequence, therefore take build pYES-GO-K-P-2 plasmid as template, based on by L115, G131, T132, V193, T353, F436, D446, Y472, I511, P535, Y537, V582, the following synthetic oligonucleotide that the mode of multiple amino acids of being replaced to M583 respectively designs and with the synthetic oligonucleotide of its complementation, use QuikChange Site-Directed Mutagensesis Kit(Stratagene company), according to appended scheme, carry out mutation operation, structure has the plasmid of sudden change glucose oxidase.

GO-L115-sudden change primer: CCACCAACAATCAGACTGCGNNNATCCGCTCCGGAAATGG(sequence number 41)

GO-G131-sudden change primer: GCTCTACCCTCGTCAACGGTNNNACCTGGACTCGCCCC(sequence number 42)

GO-T132-sudden change primer: CTCGTCAACGGTGGCNNNTGGACTCGCCCCCAC(sequence number 43)

GO-V193-sudden change primer: CATGGTATCAATGGTACTNNNCACGCCGGACCCCGCG(sequence number 44)

GO-T353-sudden change primer: CAACCTTCAGGACCAGACCNNNTCTACCGTCCGCTCAC(sequence number 45)

GO-F436-sudden change primer: GTCGCATACTCGGAACTCNNNCTCGACACGGCCGGAG(sequence number 46)

GO-D446-sudden change primer: GCCGGAGTGGCCAGTTTCNNNGTGTGGGATCTTCTGC(sequence number 47)

GO-Y472-sudden change primer: CATCCTCCGCCATTTCGCANNNGACCCTCAGTACTTTCTCAAC(sequence number 48)

GO-I551-sudden change primer: CTTATTTCGCTGGAGAGACTNNNCCCGGTGACAACCTCGC(sequence number 49)

GO-P535-sudden change primer: CCCGTACAACTTCCGCNNNAACTACCATGGTGTGGGTACTTG(sequence number 50)

GO-Y537-sudden change primer: GTACAACTTCCGCCCTAACNNNCATGGTGTGGGTACTTGCTC(sequence number 51)

GO-V582-sudden change primer: CTACGCAAATGTCGTCCCATNNNATGACGGTCTTTTATGCCATGG(sequence number 52)

GO-M583-sudden change primer: CTACGCAAATGTCGTCCCATGTTNNNACGGTCTTTTATGCCATGG(sequence number 53)

After plasmid after sudden change is imported is transformed in colibacillus DH5 α, carry out plasmid extraction, make sudden change library.Gained library is transformed into yeast saccharomyces cerevisiae INVSc1(Invitrogen company) in, the bacterium colony of growth is copied to and expressed after flat board, with plate assay, confirm to express and suddenly change importing (Fig. 5).For F436, cannot confirm the growth of mutant enzyme transformant.Should illustrate, experimental implementation is with reference to the handbook of pYES2.

Dull and stereotyped measuring method

Each color development liquid be impregnated in after the filter paper of 80mm, load on flat board, confirm color development.

50mM PIPES-NaOH（cont.0.1%Triton X-100）pH 7.020mL

10% glucose 5mL

25u/mL PO " Amano " 3(amano enzyme company) 5mL

Dianisidine (o-ジアニジ Application) 5mg

50mM PIPES-NaOH（cont.0.1%Triton X-100）pH 7.023mL

10% glucose 5mL

3mmol/L 1-methoxyl group PMS 1mL

6.6mmol/L NTB 1mL

3. the activity of liquid culture is confirmed

For the positive bacterium colony that can confirm with plate assay (measure not color development with GO, with GDH measure color development), carry out liquid culture, research GO activity and GDH activity.Should illustrate, experimental implementation is with reference to the handbook of pYES2.

Containing the phosphoric acid buffer 19mL of phenol

10% glucose 5mL

25u/mL PO " Amano " 3(amano enzyme company) 5mL

0.4g/dL 4-AA 1mL

50mM PIPES-NaOH（cont.0.1%Triton X-100）pH 7.021mL

10% glucose 5mL

3mmol/L PMS 3mL

6.6mmol/L NTB 1mL

To the culture supernatant of adding 20 μ L in each reagent 200 μ L, in 37 ℃, make it reaction.Within 10 minutes after reaction starts, measure absorbancy with 60 minutes time, by poor GO activity and the GDH activity obtained of absorbancy.For each mutant enzyme transformant, calculate the ratio (GDH activity/GO activity) of GDH activity and GO activity, compare (Fig. 6,7).Should illustrate, thering is the transformant (being expressed as pYES-GO in Fig. 6,7) of the GO that do not suddenly change, with inserting that plasmid before GO gene transforms and transformant (being expressed as pYES-2 in Fig. 6,7), GO " Amano " be expressed as GO in 2(Fig. 6,7), GDH " Amano " be expressed as FAD-GDH in 8(Fig. 6,7) as comparison other (contrast).

For assert that GDH/GO specific activity changes mutant enzyme transformant greatly, confirm that sudden change imports the aminoacid sequence (Fig. 8) of point.Based on the result shown in Fig. 6～8, determine effectively sudden change.First, for T132, and there is the replacement of T132V(from Threonine to α-amino-isovaleric acid) mutant enzyme transformant compared with, there is the replacement of T132A(from Threonine to L-Ala) the GDH/GO specific activity of mutant enzyme transformant high, therefore take T132A as effectively sudden change.For T353, two kinds of mutation T 353A and T353H in mutant enzyme transformant (5-1-5), have been assert, but owing to there being the mutant enzyme transformant (5-1-9,5-1-44) separately with T353A, therefore infer that 5-1-5 strain is the mixing of two kinds of strains, take T353A as effectively sudden change.For D446, the also supposition based on same, take D446H as effectively sudden change.In addition the sudden change V582S that, mutant enzyme transformant 12-1-49 has is also effectively sudden change.Should illustrate, aminoacid sequence and the corresponding base sequence (gene order) alone or in combination with the GO of above 4 kinds of sudden changes are listed below.

Sudden change: aminoacid sequence: base sequence

T132A: sequence number 7: sequence number 22

T353A: sequence number 8: sequence number 23

D446H: sequence number 9: sequence number 24

V582S: sequence number 10: sequence number 25

T132A and T353A: sequence number 11: sequence number 26

T132A and D446H: sequence number 12: sequence number 27

T132A and V582S: sequence number 13: sequence number 28

T353A and D446H: sequence number 14: sequence number 29

T353A and V582S: sequence number 15: sequence number 30

D446H and V582S: sequence number 16: sequence number 31

T132A, T353A and D446H: sequence number 17: sequence number 32

T132A, T353A and V582S: sequence number 18: sequence number 33

T132A, D446H and V582S: sequence number 19: sequence number 34

T353A, D446H and V582S: sequence number 20: sequence number 35

T132A, T353A, D446H and V582S: sequence number 21: sequence number 36

4. the confirmation of the substrate specificity of saltant type GO

For the enzyme (saltant type GO) with effective sudden change, the substrate specificity in research GDH activity.

50mM PIPES-NaOH（cont.0.1%Triton X-100）pH 7.021mL

10% substrate 5mL

3mmol/L PMS 1mL

6.6mmol/L NTB 3mL

To the culture supernatant 20 μ L that add mutant enzyme transformant (3-1-26,3-2-26,5-1-5,5-1-9,5-1-44,7-1-7,7-2-17,7-2-30,7-2-42,12-1-49) in each reagent 200 μ L, in 37 ℃, react.When starting latter 10 minutes and 60 minutes, reaction measures absorbancy, from the poor GDH activity of obtaining of absorbancy.By using GDH activity during each substrate to represent with the ratio of the GDH activity (100%) when take glucose as substrate.Should illustrate, take the transformant (being expressed as pYES-GO in Fig. 9) with the GO that do not suddenly change as contrast.

As shown in Figure 9, in mutant enzyme transformant, confirmed substrate specificity variation.The reactivity to wood sugar not in mutant enzyme transformant 5-1-5,5-1-9,7-1-7,7-2-17,7-2-30,7-2-42,12-1-49.The saltant type GO that these transformants have can say than existing FAD-GDH excellence not demonstrating aspect wood sugar reactive.Like this, utilize aminoacid replacement, successfully GO carry out GDHization, solved distinctive problem in existing FAD-GDH simultaneously.Should illustrate, by the sudden change for T132, be caused producing the reactivity to wood sugar, therefore enlighten the position corresponding with this sudden change in FAD-GDH and participated in the reactive possibility to wood sugar.

5. the confirmation of the effect of sudden change combination

For thinking the effectively combination of the transgenation of sudden change, verified effect.For simple purifying below, utilize PCR to react and make the C-terminal of the glucose oxidase gene from aspergillus niger GO-1 bacterium has been added to histidine-tagged GO gene (GO-3), be inserted in pYES2, build pYES-GO-3 plasmid.

(composition of reaction solution)

10 × LA damping fluid (Takara-bio Co., Ltd.), 5 μ L

2.5mM dNTPs(Takara-bio Co., Ltd.) 8 μ L

25mM MgCl2(Takara-bio Co., Ltd.) 5 μ L

Forward primer (50 μ M) 1 μ L

Reverse primer (50 μ M) 1 μ L

Template 1 μ L

LA Taq(Takara-bio Co., Ltd.) 0.5 μ L

stH2O 28.5μL

(sequence of primer)

Forward primer: GATCAGAAGCTTAAAAAAATGTCTACTCTCCTTGTGAGCTCG(sequence number 39)

Reverse primer: GATCAGCTCGAGTCAATGGTGATGGTGATGATGCTGCATGGAAGCATAATC(sequence number 54)

(reaction conditions)

Be that 94 ℃ of reactions are after 2 minutes, will be in 94 ℃ of reactions 30 seconds, in 52 ℃ of reactions 30 seconds, after 72 ℃ of reactions reaction cycle of 2 minutes repetition 35 times, in 72 ℃ of reactions 7 minutes, finally in 4 ℃ of placements.

Amplified production after PCR is inserted to pYES2, make pYES-GO-3 plasmid, confirm the sequence of inset.Sequence is not pinpointed the problems, therefore take build pYES-GO-3 plasmid as template, following synthetic oligonucleotide based on being designed to T132A, T353A, D446H, V582S to replace and with the synthetic oligonucleotide of its complementation, use QuikChange Site-Directed Mutagensesis Kit(Stratagene company), according to appended scheme, carry out mutation operation, construct the plasmid with multiple mutation glucose oxidase.

GO-T132A-1:CTCGTCAACGGTGGCGCTTGGACTCGCCCCCAC(sequence number 55)

GO-T353A-1:CCTTCAGGACCAGACCGCTTCTACCGTCCGCTCAC(sequence number 56)

GO-D446H-1:GGAGTGGCCAGTTTCCATGTGTGGGATCTTCTGC(sequence number 57)

GO-V582S-1:CGCAAATGTCGTCCCATTCTATGACGGTCTTTTATGCCATGG(sequence number 58)

After plasmid after sudden change is imported transforms in colibacillus DH5 α, carry out plasmid extraction, make sudden change library.Gained library is transformed into yeast saccharomyces cerevisiae INVSc1(Invitrogen company) in, for the bacterium colony of growth, carry out liquid culture, research GO activity and GDH activity.Should illustrate, experimental implementation is with reference to the handbook of pYES2.

Containing the phosphoric acid buffer 19mL of phenol

10% glucose 5mL

25u/mL PO-35mL

0.4g/dL 4-A.A 1mL

50mM PIPES-NaOH（cont.0.1%Triton X-100）pH 7.021mL

10% glucose 5mL

3mmol/L PMS 3mL

6.6mmol/L NTB 1mL

To the culture supernatant of adding 20 μ L in each reagent 200 μ L, in 37 ℃, make it reaction.Reaction starts to measure absorbancy with 30 minutes time in latter 10 minutes, from poor GO activity and the GDH activity obtained of absorbancy.For each mutant enzyme transformant, calculate the ratio (GDH activity/GO activity) of GDH activity and GO activity, compare.Should illustrate, to have, insert the transformant (being expressed as pYES-GO-3) of the histidine-tagged GO that do not suddenly change, be expressed as GO with transformant (being expressed as pYES-2), GO " Amano " 2(that the plasmid inserting before GO gene is transformed), GDH " Amano " 8(is expressed as FAD-GDH) be comparison other (contrast).

Result as shown in figure 11.With T132A and V582S(pYES-GO-M7), D446H and V582S(pYES-GO-M10), T132A, D446H and V582S(pYES-GO-M13) the wild enzyme (pYES-GO-3) of multiple mutation enzyme before importing with independent mutant enzyme or sudden change respectively compare, confirm that GDH/GO specific activity changes greatly.

6. the specific activity of purifying multiple mutation type enzyme and the confirmation of substrate specificity

Then, for having confirmed T132A, the D446H of effect, the combination of V582S (T132A and D446H, T132A and V582S, D446H and V582S, T132A, D446H and V582S), carry out the liquid culture of transformant, carry out after purifying research specific activity and substrate specificity with Ni-sepharose.Should illustrate, the expression in liquid culture is with reference to the handbook of pYES2.

50mM PIPES-NaOH（cont.0.1%Triton X-100）pH7.021mL

10% glucose 5mL

3mmol/L PMS 3mL

6.6mmol/L NTB 1mL

To the purifying enzyme solution or the standard enzyme solution that add 20 μ L in each reagent 200 μ L, then make it reaction in 37 ℃, obtain that from reaction, to start 5 minutes absorbancys during with 10 minutes poor.By the calibration curve of obtaining by standard enzyme, obtain activity value, calculate specific activity together with the protein content of obtaining by Bradford method.Result as shown in figure 12.The specific activity of gained purifying enzyme is about 3～8u/mg(protein).

For the enzyme with effective sudden change, use purifying enzyme, substrate specificity in research GDH activity.

50mM PIPES-NaOH（cont.0.1%Triton X-100）pH 7.021mL

10% substrate 5mL

3mmol/L PMS 3mL

6.6mmol/L NTB 1mL

For each reagent 200 μ L, add the culture supernatant 20 μ L of mutant enzyme transformant (M6, M7, M10, M13), in 37 ℃, make it reaction.Reaction starts to measure absorbancy with 10 minutes time in latter 5 minutes, by the poor GDH activity of obtaining of absorbancy.By using GDH activity during each substrate, use the ratio of the GDH activity (100%) when take glucose as substrate to represent.Should illustrate, take GDH " Amano " 8(, be expressed as FAD-GDH) be contrast.

Result as shown in figure 13.In mutant enzyme transformant, confirm that substrate specificity changes.Particularly, at D446H and V582S(pYES-GO-M10) in the reactivity to wood sugar not.The saltant type GO that this transformant has can say than existing FAD-GDH excellence not demonstrating aspect wood sugar reactive.Utilize like this aminoacid replacement, when GO carry out GDHization, successfully solved distinctive problem in existing FAD-GDH.

The amino acid whose research of optimum of 7.D446 and V582 multiple mutation enzyme

For the sudden change combination of D446 and V582, with each amino acid, become the mode of optimum amino acid combination, take pYES-GO-K-P-2 plasmid as template, synthetic oligonucleotide based on sequence number 47 and sequence number 52 and with the synthetic oligonucleotide of its complementation, use QuikChangeSite-Directed Mutagensesis Kit(Stratagene company), according to appended scheme, carry out mutation operation, build the plasmid with sudden change glucose oxidase.After plasmid after sudden change is imported is transformed in colibacillus DH5 α, carry out plasmid extraction, make D446 and V582 multiple mutation library.By gained library in yeast saccharomyces cerevisiae INVSc1(Invitrogen company) in transform, for gained transformant, use 96 hole depth orifice plates, carry out liquid culture, take implement combination before research be D446H and V582S as contrast, obtained the transformant that GDH activity and GDH/GO specific activity improve.Should illustrate, experimental implementation is with reference to the handbook of pYES2.

Result as shown in figure 14.In the combination of D446H and V582R, D446H and V582L, D446H and V582P, compared with the combination D446H and V582S that implement before research, GDH activity and GDH/GO specific activity improve.Wherein, the GO activity of D446H and V582P, below detectability, is utilized aminoacid replacement, successfully complete GDHization.

The property research of 8.D446H and V582P multiple mutation enzyme

(1) cultivation and purifying

Being expressed as follows in liquid culture carried out: with reference to the handbook of pYES2, by preserving plate, be inoculated into the 100mL making contain in 0.67% the not substratum (pH5.4) containing amino acid yeast nitrogen (Nippon Becton Dickinson Co., Ltd. system), 2% glucose in 500mL shaking flask (slope mouth Off ラスコ), carry out the preculture of 30 ℃, 140 revs/min, 20 hours.

After preculture finishes, by centrifugal thalline recovery, again with what become that the mode of OD660=0.4 is inoculated into the 100mL that makes in 500mL shaking flask, contain 0.67% not containing in the substratum (pH5.4) of amino acid yeast nitrogen (Nippon Becton Dickinson Co., Ltd. system), 2% semi-lactosi and 1% raffinose, carry out this cultivation of 30 ℃, 140 revs/min, 5 hours.

After cultivation finishes, utilize ultra-filtration membrane (trade(brand)name MICROZA, grouping molecular weight 6000, society of Asahi Chemical Industry system) carry out desalination and concentration, for above-mentioned desalination and concentration liquid, make it to adsorb in anionite-exchange resin (trade(brand)name HiTrap DEAE FF, the GE Healthcare Japan company system) post with 20mM phosphoric acid buffer (pH7.0) equilibration, clean with above-mentioned damping fluid.After cleaning, use the 30mM MOPS damping fluid (pH7.0) that contains NaCl, utilize the Liner gradient method of NaCl concentration 0～1.0M to carry out stripping.Can obtain by the partially purified saltant type GO(D446H of purification process as above V582P multiple mutation enzyme).

(2) confirmation of substrate specificity

For saltant type GO partially purified enzyme (D446H, V582P multiple mutation enzyme), the substrate specificity in research GDH activity.

50mM PIPES-NaOH（cont.0.1%Triton X-100）pH 7.021mL

10% substrate 5mL

3mmol/L PMS 3mL

6.6mmol/L NTB 1mL

For each reagent 200 μ L, add saltant type GO partially purified enzyme (D446H, V582P multiple mutation enzyme) 20 μ L, in 37 ℃, make it reaction.Reaction starts to measure absorbancy with 30 minutes time in latter 10 minutes, by the poor GDH activity of obtaining of absorbancy.By using GDH activity during each substrate, use the ratio of the GDH activity (100%) when take glucose as substrate to represent.Should illustrate, to be expressed as FAD-GDH in GDH " Amano " 8(Fig. 6,7) be contrast.

Result as shown in figure 15.Saltant type GO partially purified enzyme (D446H, V582P multiple mutation enzyme) does not demonstrate the reactivity to wood sugar, and compared with GDH " Amano " 8, substrate specificity is especially excellent.

In industry, utilize possibility

The detection of the glucose amount of sudden change provided by the invention GO in sample, useful in quantitatively.On the other hand, method of design of the present invention, preparation method are utilized as obtaining the method that GDH that characteristic improves or GO obtain.Particularly, expect as utilizing for the method that obtains the GO of GDHization or the GDH of GOization.

The present invention is not limited to the explanation of any foregoing invention embodiment and embodiment.In the situation that not departing from invention which is intended to be protected, the various variant in the scope that those skilled in the art can easily expect are also contained in the present invention.

The contents such as paper, publication communique and the patent gazette expressed in this specification sheets are quoted by quoting its full content.

Another attached sequence list text none

Sequence number 39～40,54: the explanation of artificial sequence: PCR primer

Sequence number 41～53,55～58: the explanation of artificial sequence: sudden change importing primer

Claims

1. a mutant enzyme, is consisted of the arbitrary aminoacid sequence in sequence number 7～21,59～61.

2. a gene, the mutant enzyme claimed in claim 1 of encoding.

3. gene according to claim 2, consists of the arbitrary base sequence in sequence number 22～36,62～64.

4. a recombinant DNA, comprises the gene described in claim 2 or 3.

5. a microorganism, has recombinant DNA claimed in claim 4.

6. a methods for dextrose, is characterized in that, right to use requires the mutant enzyme described in 1, measures the glucose in sample.

7. a glucose assays reagent, is characterized in that, comprises mutant enzyme claimed in claim 1.

8. a glucose assays test kit, comprises glucose assays reagent claimed in claim 7.

9. a method, is characterized in that, right to use requires the mutant enzyme described in 1, reduces the glucose amount in mechanicals or its raw material.

10. an enzyme agent, contains mutant enzyme claimed in claim 1.

The method of design of 11. 1 kinds of mutant enzymes, comprises the following steps (i) and (ii):

(i) in the aminoacid sequence of sequence number 1, determine one or more the amino acid whose step being selected from following (3), (5), (7), (12):

(ii) the aminoacid sequence based on sudden change object enzyme, be structured in step in (i) definite aminoacid sequence by other aminoacid replacement and sequence number 7～21,59～61 in the step of arbitrary aminoacid sequence.

12. method of design according to claim 11, wherein, step (i) in substituted amino acid for amino acid, the amino acid of (7) or the amino acid of (12) of (3) or be selected from the plural amino acid whose combination in them.

13. method of design according to claim 11, wherein, step (i) in substituted amino acid be the amino acid of (7) and the amino acid of (12).

The preparation method of 14. 1 kinds of mutant enzymes, comprise the following steps (I)～(III):

(I) step of the nucleic acid that the arbitrary aminoacid sequence in sequence number 7～21,59～61 is encoded in preparation;

(II) step that described nucleic acid is expressed, and

(III) step of recovery expression product.